The trans-complementation of a replication defective human immunodeficiency, type 1 (HIV-1), art/trs negative mutant provirus by the art/trs protein was characterized by amplified viral replication. The enhanced trans-activation was unique to the art/trs mutant, whose phenotype was distinguished by accumulation of HIV-1 B(3')-ORF and tat proteins and virtual lack of viral structural protein synthesis. The virus produced by trans- complementation was not infectious for CD4+ lymphocytes and the rescued phenotype could not be passaged by cocultivation of transfectants with CD4+ lymphocytes thus eliminating the possibility that the mutation was rescued by recombination. The magnitude of transactivation of mutant provirus was relatively constant over a wide range of art/trs expressing cDNA (0.5-20 mug). art/trs protein was also absolutely required for the transient expression of env gp160 from subgenomic plasmids containing HIV env ORF (in the spliced or presliced configuration) fused to either HIV LTR or an heterologous promoter. The 3'(B)-ORF product of the Human Immunodeficiency Virus type 1 (HIV-1) was shown to be a negative regulator of HIV-1 replication. B ORF mutant proviruses replicated more vigorously than their standard countparts during the transient expression in a variety of nonlymphoid and lymphoid cell lines. The B ORF product supplied in trans as an expressible cDNA, suppressed in a dose-dependent manner, the standard replication of wild type provirus and the enhanced replication of B ORF mutant proviruses. Transient expression of reporter genes that were linked to the HIV LTR was also repressed by coexpression of B ORF. The negative effect of B ORF was mediated by a concentration dependent inhibition of transcription from HIV-1 LTR containing a to be negative regulatory element, located between 340 and 156 nucleotides upstream of the RNA initiation site.